Characterization of protein cross-links via mass spectrometry and an open-modification search strategy.
Protein-protein interactions are key to function and regulation of many biological pathways. To facilitate characterization of protein-protein interactions using mass spectrometry, a new data acquisition/analysis pipeline was designed. The goal for this pipeline was to provide a generic strategy for identifying cross-linked peptides from single LC/MS/MS data sets, without using specialized cross-linkers or custom-written software. To achieve this, each peptide in the pair of cross-linked peptides was considered to be "post-translationally" modified with an unknown mass at an unknown amino acid. This allowed use of an open-modification search engine, Popitam, to interpret the tandem mass spectra of cross-linked peptides. False positives were reduced and database selectivity increased by acquiring precursors and fragments at high mass accuracy. Additionally, a high-charge-state-driven data acquisition scheme was utilized to enrich data sets for cross-linked peptides. This open-modification search based pipeline was shown to be useful for characterizing both chemical as well as native cross-links in proteins. The pipeline was validated by characterizing the known interactions in the chemically cross-linked CYP2E1-b5 complex. Utility of this method in identifying native cross-links was demonstrated by mapping disulfide bridges in RcsF, an outer membrane lipoprotein involved in Rcs phosphorelay.
Singh
Pragya
P
Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, USA.
Shaffer
Scott A
SA
Scherl
Alexander
A
Holman
Carol
C
Pfuetzner
Richard A
RA
Larson Freeman
Theodore J
TJ
Miller
Samuel I
SI
Hernandez
Patricia
P
Appel
Ron D
RD
Goodlett
David R
DR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D020134:Catalytic Domain; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry / Q000378:metabolism; D015786:Cytochromes b5 / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D015203:Reproducibility of Results
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; D010455:Peptides; D011506:Proteins; D015786:Cytochromes b5; D019392:Cytochrome P-450 CYP2E1